Method of preparing an artificial tooth primordium in vitro and artificialtooth primordium derived therefrom

ABSTRACT

The present invention is directed to a method of preparing an artificial tooth primordium in vitro, comprising the steps: a) providing isolated mesenchymal dental pulp cells; and b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium; as well as to an artificial tooth primordium derived therefrom.

BACKGROUND OF THE INVENTION

Dental care is the best way of maintaining healthy denture. However,even under optimal dental care impairment, destruction or loss of teethcan occur. Such impairment, destruction or loss of teeth may be e.g.disease-related, hormone-related, therapy-related or a consequence of anaccident. Nowadays, in such a case dental implants are used whichusually are formed of materials comprising metal (e.g. titan), ceramicsand/or composites. These implant techniques are highly sophisticated andfrom a medical and cosmetic point of view achieve already high levels.However, these tooth replacement therapies require a significantsurgical procedure and, eventually, laborious follow-up care. Inaddition, these synthetic implants do not meet all demands on a toothlike e.g. an “absorption” by the natural periodontal ligament whichfixes the tooth within the jawbone.

Thus, it is an aim in the field of regenerative medicine to providemeans which allow the production or induction of tissue or organs thatequal the respective naturally occurring tissues and organs in functionand morphology. One approach is the provision of artificial tissue ororgans that can be used in tooth regeneration in vitro and/or in vivo.Such artificial tissue comprises tissue that has been produced bybiotechnological means.

Apart from the problem of providing a tissue that is capable of forminga tooth in vivo that sufficiently equals naturally occurring teeth infunction and morphology, it is another object to provide a tissue thatcan be manufactured without the need of embryonic stem cells and thatmakes use of cells originating from the patient to be treated.

In numerous approaches first attempts and progress have been madetowards tissue that serves this purpose. Examples of such approaches aredescribed in EP 1 905 459 A1, EP 2 130 909 A1, US 2007/0231275 A1, US2011/0212414 and WO 2005/051436 A2. However, in most of these approachesembryonic cells are used, the cells are cultured under non-physiologicaladherent culture conditions and require the presence of a syntheticscaffold or carrier.

Thus, it is an option of the present invention to provide means toovercome one or more problems of the prior art. In particular it is anoption of the invention to provide an improved method of preparing anartificial tooth primordium in vitro, which can be performed withnon-embryonic primary cells.

DISCLOSURE OF THE INVENTION

The present invention relates to a method of preparing an artificialtooth primordium in vitro, comprising the steps of:

a) providing isolated mesenchymal dental pulp cells; and

b) culturing the mesenchymal dental pulp cells under non-adherentconditions to form a cell aggregate representing an artificial toothprimordium.

It has surprisingly been found that isolated mesenchymal dental pulpcells are capable of forming a three dimensional cell aggregaterepresenting an artificial tooth primordium without influence ofembryonic epithelial cells. This effect is achieved by culturing themesenchymal dental pulp cells under non-adherent conditions. It could beshown that under such non-adherent culture conditions the mesenchymaldental pulp cells arrange freely to each other and condense to a cellaggregate which exhibits expression of markers specific for tooth germstructures and which are therefore denoted tooth primordium.

In the following, different aspects of the invention are provided inmore detail. Each aspect so defined may be combined with any otheraspect or aspects unless clearly stated to the contrary. In particular,any feature indicated as being preferred or advantageous may be combinedwith any other feature or features indicated as being part of theinvention or being indicated as being preferred or advantageous.

The method of the present invention is directed to the preparation of anartificial tooth primordium in vitro. An artificial tooth primordium isa tooth primordium that has been prepared and assembled de novo and invitro.

For the purpose of the present invention the term “tooth primordium”refers to a functional cell aggregate of one or more than one cell typethat exhibits at least one function that is specific for dental tissueor a functional tooth germ. Preferably the tooth primordium of thepresent invention exhibits the majority or essentially all organ ortissue functions of a dental tissue or a functional tooth germ or tooth.In particular the tooth primordium of the present invention may behavelike a functional inductive tooth germ and is capable of inducing toothorgan development or development of a complete tooth in vitro and/or invivo. The tooth primordium of the present invention may be characterizedby elevated expression of marker genes or proteins that are associatedwith development or differentiation of dental tissue. Preferably thetooth primordium of the present invention may be characterized byelevated expression of BMP4, HGF, PAX9, MSX1, Collagen type I, DSPPand/or predentin compared to isolated mesenchymal dental pulp cellsimmediately prior to subjecting to non-adherent culturing conditions.Furthermore, a basement membrane formation, mainly characterized byCollagen type IV expression, may occur in the aggregate. Aggregateformation and culturing conditions may lead to a mineralization of theresulting tissue.

The artificial tooth primordium of the present invention is formed by amethod of the present invention. The artificial tooth primordium mayexhibit a cell aggregate with a substantially spherical shape and withan average diameter of 0.3 mm to 2 mm, preferably with an averagediameter of 0.5 mm to 1.5 mm. The artificial tooth primordium maycomprise a cell aggregate comprising an inner core formed of mesenchymaldental pulp cells and an outer layer formed of epithelial cells whereasthe epithelial cells may invaginate the inner core at later points intime of co-culturing. Invagination of the cells of epithelial origin maylead to a ameloblastic differentiation of these cells. Thisdifferentiation may be characterized by the morphological appearance(columnar arrangement) as well as by elevated expression of ameloblastmarker genes or proteins (e.g. BMP7, SHH and/or amelogenin). Theartificial tooth primordium of the invention is free of any artificialbiological or non-biological scaffold or carrier which does notoriginate from the cells used in the production of said artificial toothprimordium. The artificial tooth primordium of the invention preferablyconsists of the cell aggregate formed by a method of the invention,wherein the method has been conducted without the use of any artificialbiological or non-biological scaffold or carrier which does notoriginate from the cells used in said method.

In the method of the invention, isolated mesenchymal dental pulp cellsare used. The term “isolated” means that the mesenchymal dental pulpcells are cells that have been isolated from a natural source or progenythereof which e.g. has been derived by cell proliferation. Themesenchymal dental pulp cells used are preferably derived from dentalpulp tissue of a donor tooth or tissue. Preferably, the mesenchymaldental pulp cells are primary cells which have not been transformed orimmortalized. These cells may be characterized by positivity for CD90,CD73, CD44, CD29 and HLA I and negativity for CD34 and CD45. Theyexhibit a spindle-shaped morphology in 2D culture and adhere to plastic.In particular, the mesenchymal dental pulp cells may comprise or consistof adult mesenchymal dental pulp cells. Such adult mesenchymal dentalpulp cells are derived from non-embryonic dental pulp tissue of a donortooth or tissue. The mesenchymal dental pulp cells can be derived fromdental pulp tissue of any donor tooth or tissue that has beendifferentiated to comprise dental pulp tissue. Preferably themesenchymal dental pulp cells are derived from dental pulp tissue of atooth, more preferably from a third molar of a donor. The mesenchymaldental pulp cells used in the method of the invention are preferablyhuman dental pulp cells. Human mesenchymal dental pulp cells are derivedfrom dental pulp tissue of human origin.

In the method of the invention, the mesenchymal dental pulp cells can besubjected to culturing under non-adherent conditions at any stage afterisolation. However, the formation of cell aggregates is further enhancedif the mesenchymal dental pulp cells have undergone at least someculturing under adherent 2D monolayer conditions. Preferably, themesenchymal dental pulp cells have been cultured in 2D monolayer culturefor at least 1 passage prior to subjecting to culturing undernon-adherent conditions. A high efficiency in formation of cellaggregates is maintained over a broad spectrum of passages. However, ithas been found that best results are achieved if the mesenchymal dentalpulp cells have been cultured in 2D monolayer culture for at least 1passage and not more than 15 passages. Preferably the mesenchymal dentalpulp cells are subjected to non-adherent culture after culture in 2Dmonolayer for at least 2 passages and not more than 8 passages afterisolation.

In the method of the invention, after provision of the isolatedmesenchymal dental pulp cells, the mesenchymal dental pulp cells aresubjected to non-adherent culture conditions to form cell aggregates.

The formation of cell aggregates is particularly effective if themesenchymal dental pulp cells are subjected to non-adherent cultureconditions at a certain concentration. If the concentration is too low,cells have only rarely contact to each other and condensation to cellaggregates is less effective. On the other hand, if the concentration ofmesenchymal dental pulp cells is too high, cells are less flexible ormobile and, thus, the formation of cell aggregates is less effective.Preferably, the mesenchymal dental pulp cells are subjected tonon-adherent culture conditions at a concentration of 5×10⁴ to 5×10⁷ perml. Even better results are achieved if the mesenchymal dental pulpcells are subjected to non-adherent culture conditions at aconcentration of 1×10⁵ to 1×10⁷ per ml, preferably of 5×10⁵ to5×10⁶ perml, and most preferably of 9×10⁵ to 1.1×10⁶ per ml.

In the method of the invention, the isolated mesenchymal dental pulpcells are cultured under non-adherent culture conditions. This meansthat mesenchymal dental pulp cells are cultured under conditions whereinthe cells do not adopt a flattened, spread-out shape indicating strongattachment and adherence to the culture surface. Preferably themesenchymal dental pulp cells remain rounded directly after seeding and,if at all, only weakly associated with the culture surface. Appropriatemeans for non-adherent cell culture are well known in the art.Non-adherent culture conditions may comprise culture of the mesenchymaldental pulp cells in culture vessels with a culture surface that doesnot support adherence of the mesenchymal dental pulp cells. E.g. culturevessels with culture surfaces exhibiting ultra-low cell attachment canbe used. For that purpose culture vessels with a neutral or positivelycharged culture surface may be used. Preferably, the culture surface maybe coated by a layer of a material which further reduces interaction ofthe mesenchymal dental pulp cells and the culture surface. The culturesurface may be covered with a hydrophilic hydrogel.

It appears that under non-adherent culture conditions, the mesenchymaldental pulp cells associate and condense to cell aggregates ratherquickly. Already after 24 hours of non-adherent cell culture, themesenchymal dental pulp cells aggregated into one large complex.However, in order to prepare cell aggregates that represent artificialtooth primordial with progressed development, differentiation and/orfunction, it is beneficial to conduct non-adherent culturing for aperiod of time that exceeds 24 hours. In the method of the invention,the mesenchymal dental pulp cells may be cultured under non-adherentconditions for at least 48 hours, preferably for at least 72 hours, morepreferably for at least 2 weeks, even more preferably for at least 4weeks, most preferably for at least 8 weeks. Thus, the mesenchymaldental pulp cells may be cultured under non-adherent conditions for 48hours to 10 weeks, preferably for 1 week to 9 weeks, more preferably for4 weeks to 8 weeks.

In the method of the invention, the mesenchymal dental pulp cells arepreferably cultured under non-adherent conditions at least until a cellaggregate is formed which exhibits a rounded shape with an averagediameter of 0.3 mm to 2 mm, more preferably with an average diameter of0.5 mm to 1.5 mm.

The suitability of the artificial tooth primordium generated by themethod of the present invention is dependent on its ability to induce orprovide differentiated dental tissue. Thus, it is preferred to culturethe isolated mesenchymal dental pulp cells under non-adherent conditionsuntil the resulting cell aggregate starts to exhibit properties and/orfunctions of partially or fully differentiated dental tissue. Theprogression in differentiation can be monitored by the relativeexpression of respective marker genes, proteins or structures.Preferably, the isolated mesenchymal dental pulp cells are culturedunder non-adherent conditions at least until the cell aggregate formedexhibits upregulated expression of BMP4, HGF, PAX9, MSX1 Collagen typeI,DSPP and/or predentin compared to isolated mesenchymal dental pulpcells immediately prior to subjecting to non-adherent culturingconditions. Preferably, the formed aggregate exhibits characteristics ofthe so called cap stage of tooth development with respect to morphologyand gene expression. Relative expression of genes can easily bedetermined by well known methods like e.g. quantitative orsemi-quantitative RT-PCR, Transcriptome Sequencing (RNA-seq) or Northernblotting. Relative expression of proteins can also be determined by wellknown methods like e.g. Western blotting, Suspension Array Technologyand ELISA techniques. Localization of differential expression ofproteins can be determined by in sit hybridization and/orimmunohistochemistry.

One of the drawbacks encountered with most of the methods of the priorart is that in these methods, the presence of an artificial biologicalor non-biological scaffold or carrier is needed on or within which cellsare cultured to form a tooth primordium or tooth germ. A biological ornon-biological scaffold or carrier is regarded as artificial if saidscaffold or carrier is added or provided from the outside and is notformed by the cells used in the method of the invention during formationof cell aggregates. In the method of the present invention, the use orpresence of such an artificial biological or non-biological scaffold orcarrier is not needed. The method of the invention yields an artificialtooth primordium according to the invention without the use of any suchartificial biological or non-biological scaffold or carrier which doesnot originate from the cells used in the production of said artificialtooth primordium. In a preferred embodiment, the method of the presentinvention is performed such that no artificial biological ornon-biological scaffold is used in the formation of cell aggregates.

The method of the present invention is particular advantageous becauseartificial tooth primordial of the present invention can be producedstarting from a single cell type, namely starting from isolatedmesenchymal dental pulp cells. However, the generation of even moresophisticated artificial tooth primordial can be achieved if the methodfurther comprises a step of co-culturing pre-condensed mesenchymaldental pulp cells with epithelial cells. The co-culturing is performedpreferably under non-adherent culturing conditions. It appears to beparticularly beneficial to culture the isolated mesenchymal dental pulpcells under non-adherent conditions for at least 48 hours to formpre-aggregated mesenchymal dental pulp cells, then to add the epithelialcells, and to co-culture the mixture of pre-aggregated mesenchymaldental pulp cells and epithelial cells under non-adherent conditions.Co-culturing may be performed for the periods of time defined andproposed above for non-adherent culturing of isolated mesenchymal dentalpulp cells.

Preferably, the epithelial cells used in the method of the invention areprimary epithelial cells, particularly preferred human primaryepithelial cells. The primary epithelial cells may be derived from adultor non-embryonic tissue like e.g. skin tissue or gingival tissue. In apreferred embodiment the epithelial cells are keratinocytes, e.g. humankeratinocytes, more preferably primary keratinocytes derived fromnon-embryonic tissues like e.g. skin or gingiva. Particularly preferredare human keratinocytes derived from gingiva e.g. from non-embryonicgingiva.

In the method of the invention with the co-culturing step, theepithelial cells are added for co-culture such that the number ofmesenchymal dental pulp cells initially used to form pre-aggregatedmesenchymal dental pulp cells is equal to or higher than the number ofepithelial cells. Preferably, epithelial cells are added for co-culturein a relative amount of 1:1 to 1:10 related to the initial cell numberof mesenchymal dental pulp cells, preferably in a relative amount of 1:2to 1:8, more preferably in a relative amount of 1:3 to 1:5.

The method of the invention with the co-culturing step yields artificialtooth primordial of the invention, wherein the early cell aggregateforming the tooth primordium comprises a core formed of aggregatedmesenchymal dental pulp cells and an outer layer formed of theepithelial cells. The long-term cultured aggregate may exhibit aninvagination of the epithelial cells into the inner core of mesenchymalcells.

In the method of the invention, the mesenchymal dental pulp cells arecultured in adherent or non-adherent conditions with standard medium.There is no specialized culture medium necessary to induce proper cellaggregation under non-adherent conditions. The skilled person is wellaware of suitable media. Typically standard DMEM is used with a certaincontent of fetal calf serum (FCS), preferably FCS is present in aconcentration of 5% to 15%, more preferably in a concentration of 10%FCS. In case of co-culturing pre-aggregated mesenchymal dental pulpcells and epithelial cells under non-adherent conditions, the culturemedium may comprise a certain amount of culture medium usually used inculturing the epithelial cells under adherent conditions.

The present invention is also directed to a transplant comprising orconsisting of an artificial tooth primordium of the present invention orof a tissue or structure derived therefrom.

In another aspect of the invention a pharmaceutical composition isprovided comprising an artificial tooth primordium of the presentinvention, a tissue or structure derived therefrom or a transplant ofthe invention and at least one pharmaceutically acceptable excipient.

The artificial tooth primordium of the invention, the transplant of theinvention or the pharmaceutical composition of the invention may be usedin the treatment of dental impairment and/or destruction or loss ofteeth.

Since the method of the invention works with isolated mesenchymal dentalpulp cells derived from non-embryonic sources, the method allows theproduction of artificial tooth primordia starting from cells derivedfrom a particular donor or patient. Thus, it is possible to provideartificial tooth primordia that have been derived from the cells of theperson to be treated with the artificial tooth primordium,pharmaceutical composition or transplant of the invention. Thus, itappears possible to provide a transplant that is mainly, substantiallyor completely derived from cells of the recipient of the transplanthimself so that rejection reactions will be reduced to a minimum or willbe completely absent.

The artificial tooth primordium of the invention may be used for the invitro generation of tooth tissue or whole teeth.

The artificial tooth primordium of the invention, the transplant of theinvention or the pharmaceutical composition of the invention may be usedas a research tool that can be used in vitro and in vivo.

The artificial tooth primordium of the invention, the transplant of theinvention or the pharmaceutical composition of the invention may be usedin a method of screening for substances in vitro or in vivo, whichmodulate properties of tooth tissue.

The present invention additionally teaches a method for screeningsubstances in vitro, which modulate properties of tooth tissue,comprising the steps of:

-   -   providing a sample of an artificial tooth primordium of the        invention or a cell aggregate prepared by a method of the        invention or a tissue derived therefrom;    -   dividing the respective sample into portions;    -   incubating at least one portion with a substance to be screened;        and    -   comparing parameters measured for the treated portion with        another portion that was not incubated with the substance to be        screened.

In a preferred embodiment, the portion is subjected to a self-containedcirculation system prior to incubating the portion with a substance tobe screened.

Briefly, the inventive method makes the identification and analysis ofsubstances possible, which exert an influence on tooth or tooth tissuevia the artificial tooth primordium of the invention. The sample, whichshall be understood to comprise a certain number of product subjectsaccording to the invention, is divided into multiple portions. At leasttwo subsets are provided; one is used for screening while the other oneserves as negative control. Preferably, the number of screening partsexceeds the number of control parts. Usually, numerous portions aresubjected to a high-throughput screening. The substances to be screenedin the inventive method are not restricted anyway. In an embodiment ofthe invention, the substances are selected from the group of nucleicacids including RNAi, ribozymes, aptamers, antibodies, peptides,carbohydrates, polymers, small molecules having a molecular weightbetween 50 and 1,000 Da, and proteins, preferably antibodies, cytokinesand lipocalins. These substances are often available in libraries. It ispreferred to incubate a single compound within a distinct portion of thesample. However, it is also possible to investigate the cooperativeeffect of substances by incubating at least two substances within oneportion. A further subset of subjects is simultaneously incubated in theabsence of the substances. The incubation process depends on variousparameters, e.g. the cell types and the sensitivity of detection, whichoptimization follows routine procedures known to those skilled in theart. The identification of effective substances in the meaning of theinvention is preferably indirectly performed, e.g. by determining theexpression patterns and/or the cell viability, which are altered. Thedetermination may be performed at a specified moment and correlated tothe signal strength at the beginning of the experiment and the negativecontrol. Suitable tests are known to those skilled in the art or can beeasily designed as a matter of routine.

Since the artificial tooth primordium of the invention can be regardedas an organ or a precursor of an organ or part thereof, it may beparticularly beneficial to use a test system wherein the artificialtooth primordium can be prepared and/or cultured for a prolonged timeunder conditions which mimic natural perfusion. It appears particularlysuitable to combine the method of the present invention and/or theartificial tooth primordium of the present invention in an assay systembased on the self-contained organ-on-a-chip device described in theEuropean patent application with the filing number EP 10 008 244 or inthe PCT application with the publication number WO 2009/146911 A2.

The present invention provides for the first time an artificial toothprimordium and a method of producing the same, which is characterized bythe lack of requirement of any artificial biological or non-biologicalscaffold or carrier, which is produced from adult cells so that noembryonic cells or tissues are needed and which requires only cells ofone cell type, namely isolated mesenchymal dental pulp cells, or cellsof two cell types, namely isolated mesenchymal dental pulp cells andkeratinocytes.

The artificial tooth primordium of the present invention represents afunctional inductive tooth germ which is capable of tooth organdevelopment in vitro and in vivo. The resulting tooth organ ischaracterized by the verifiable presence of structures usually formingpart of the developing tooth which are arranged in the physiologicalorder of a tooth:

-   -   enamel, i.e. ameloblasts;    -   dentin;    -   dental pulp, incl. odontoblasts and undifferentiated dental pulp        cells;    -   cementum, i.e. cementoblasts;    -   periodont; and    -   alveolar bone.

FIGURES

FIG. 1 shows three-dimensional low-attachment culture of humanmesenchymal dental pulp cells; scale bars: 500 μm.

FIG. 2 shows human dental pulp cells in co-culture with keratinocytes;scale bars: 500 μm. Given time points indicate hours after addition ofkeratinocytes.

FIG. 3 shows self-assembled cell type compartmentalization of theprimordium described by the invented method; scale bars: A: 100 μm B,C:500 μm D: 300 μm. Given time points indicate time after addition ofkeratinocytes.

FIG. 4 shows expression of intra- and extracellular molecules tocharacterize the constructed tooth primordium.

EXAMPLES

General Methods

Mesenchymal Dental Pulp Cell Isolation

Dental pulp cell isolation is performed according to a modified protocolfrom Gronthos et al. (A method to isolate and culture expand humandental pulp stem cells. Methods Mol Biol. 2011; 698:107-21).

-   -   1 Extracted third molars from patients after informed consent        are collected in DMEM containing 10% FCS and penicillin and        streptomycin (100 μg/ml each) and stored at 4° C. for not longer        than 24 hours.    -   2 The biopsies are handled under sterile conditions throughout        the whole procedure of cell extraction. Tooth crown and the root        if present are wiped with 100% ethanol. To open the pulpal        cavity, the tooth is split by mechanical cracking with a hammer.    -   3 Pulp tissue is removed with forceps and placed into a PBS        containing petri dish.    -   4 The tissue is then cut into small fragment which are then        washed twice with PBS to remove debris and blood.    -   5 Afterwards a digestion step is performed with a collagenase (3        mg/ml)/dispase II (4 mg/ml) enzyme mix for 2 h at 37° C.    -   6 The digested solution is the filtered through a 70 μm cell        strainer and washed twice with PBS by centrifugation (400×g for        5 min). The remaining cell pellet is resuspended in DMEM w/10%        FCS and penicillin and streptomycin (100 μg/ml each).

Cell Culture

Culture and expansion of mesenchymal dental pulp cells is achieved bymaintaining cells in monolayer culture onto culture flask surfaces foradherent cells in DMEM w/10% FCS and penicillin and streptomycin (100μg/ml each) under standard culture conditions (5% CO2, 37° C.). Theadherent fibroblast-like mesenchymal cells are grown to 90% confluencyand then passaged.

Aggregation/Condensation Process for Formation of Artificial ToothPrimordium

For culture under non-adherent conditions, mesenchymal dental pulp cellsof approx. 2^(nd) to 8^(th) passage are passaged two days prior to use,were harvested and resuspended in DMEM+10% FCS to yield up in a singlecell suspension of 106 cells per ml. The cell suspension (1 ml per well)was given to 24 well low attachment plate (Ultra Low Cluster Plate,Corning, Germany). In contrast to the negatively charged, hydrophilicsurface of standard tissue culture dishes the ultra low attachmentplates possess a neutral, hydrophilic hydrogel coated surface thatgreatly minimizes the binding of attachment proteins. By using thisspecialized culture dishes the mesenchymal dental pulp cells do notsettle down through cell adhesion as in micromass culture. The formationof a 2D monolayer culture was prevented and the cells retained a roundshape under this suspension culture maintaining non-adherent conditions.Furthermore, in contrast to methods relying on an external scaffold orcarrier, the low attachment culture system provides the opportunity forfree cell movement and cell cell interaction during an initialcondensation process. Condensation process starts shortly after seedingand is observed macroscopically by cells forming aggregates. To ensureconstant culture conditions medium was changed regularly every 3 days.

Co-Culture

If co-culture of mesenchymal dental pulp cells and cells of epithelialorigin (skin derived or gingival keratinocytes) is intended, thecondensates produced described by the method above are transferred atday 2 to 5 in a composite medium appropriate for both cell types (e.g.standard DMEM (w/FCS) and Keratinocyte Culture Medium; 1:1). A singlecell suspension of epithelial cells in a ratio of 1:4 related to theinitial cell number used for mesenchymal condensation was added and theresulting mixture was cultured under non-adherent conditions. To ensureconstant culture conditions medium was changed regularly every 3 days.

Results

In order to expand human dental pulp cells (hDPCs) used for preparationof an artificial tooth primordium in vitro, the isolated cells werecultured for at least 2 passages in monolayer. As described cells adopta fibroblastic morphology and express MSC surface markers (CD90, CD73,CD44, CD29 and HLA I (data not shown). To accomplish an induction ofcellular arrangement and differentiation, hDPCs are cultured in athree-dimensional manner. The idea of the low-attachment culturetechnique described above is to avoid cell attachment to the culturedish surface and to allow self-organization mediated by cell mobility.Therefore, human mesenchymal dental pulp cells (hDPCs) were seeded in adensity of 1×10⁶ cells/ml in low-attachment culture plates in normalDMEM with 10% FCS and were cultured at standard cell culture conditions.Dental pulp cells arrange freely to each other and condense to athree-dimensional cell aggregate. Condensation begins quickly afterseeding. Already after 4 hours most of the cells are associated to forma cell aggregate. Within 24 hours after seeding, the cell aggregatefurther condenses (FIG. 1). This cell aggregate remains viable for aprolonged period of time and further develops and differentiates into anartificial tooth primordium of the present invention with an average of0.3 mm in diameter.

To assess whether these mesenchymal condensated are capable ofinteraction with human gingival keratinocytes and whether anameloblastic differentiation can be induced by these condensates hDPCcondensates and keratinocytes were co-cultured in the three-dimensionallow-attachment system. Here, primary human keratinocytes isolated fromgingiva were added as single cell suspension of epithelial cells in aratio of 1:4 related to the initial cell number used for mesenchymalcondensation to 48 h old mesenchymal hDPC aggregates. The resultingmixture was then cultured under non-adherent conditions. Keratinocytesarrange around the three-dimensional mesenchymal aggregate. The gingivalkeratinocytes are attracted and rapidly assemble around the condensated(FIG. 2).

During tooth development a strong interaction between the mesenchymaland epithelial tissues is observed leading to proliferation andinvagination. In order to trace the interplay between these two celltypes induced by the culture method described by the invention, the usedcells were fluorescently labelled. Constitutively eGFP-expressing hDPCswere seeded in a density of 1×10⁶ cells/ml in low-attachment cultureplates and cultured as described above for 24 hours. Afterwards primaryhuman keratinocytes tracked with CellTracker™ Red CMTPX (MolecularProbes®) were added as single cell suspension of epithelial cells in aratio of 1:4 related to the initial cell number used for mesenchymalcondensation and the resulting mixture was cultured under non-adherentconditions. After three days, keratinocytes are attached to the surfaceof the mesenchymal aggregate (FIG. 3A). Four weeks of co-culture lead toa completely “interwoven” structure within the self-assembledcondensate. At this point in time keratinocytes can be detectedunderneath a layer of hDPCs within the constructed organoid byfluorescence microscopy (FIG. 3B). This observation is further evidencedby fluorescent microscopic analysis of cryo-sections (8 μm thickness)(FIG. 3C). Combined hematoxylin/eosin staining further elucidates thestructural compartmentalization of the artificial tooth primodium (FIG.3D). Keratinocytes which appear distinctly pink due to expression ofacidic proteins (e.g. Cytokeratin 15 and 18) invaginated at one site ofthe three-dimensional condensate. The band of epithelial tissue whichconnects the invaginated cells with the surrounding epithelium resemblesthe dental lamina during tooth development. The mesenchyme is clearlyseparated from the outer epithelium by a connective tissue-like layerand completely surrounds the invaginated structure.

Artificial tooth primordia prepared by the method of the invention weresubjected to comprehensive immunohistological analysis after 8 weeks ofculture. The constructed organoids were characterized regarding theexpression of intra- and extracellular molecules. For detection of therespective proteins, primary antibodies were used against Vimentin,Cytokeratin 15, Collagen type I, Collagen type IV and Ki67. Secondaryantibodies were coupled with Alexa Fluor® 594 (red) for detection underfluorescence microscope (FIG. 4). All sections were counterstained withHoechst33258 (blue). Vimentin (FIG. 4A/A′) and Cytokeratin 15 (FIG. 4B)clearly mark the areas of mesenchyme and epithelium respectively.Collagen type I (FIG. 4C) is exclusively expressed in the mesenchymalpart of the aggregate as part of extracellular matrix. Note thatCollagen type I is a major component of dentin. Collagen type IV (FIG.4D) is highly expressed at the interface of the two cell types mimickinga basal lamina. Proliferative activity is exclusively detected in theepithelial cell layers as indicated by the presence of Ki67 (FIG. 4E).Note the columnar arrangement of the invaginated epithelium (FIG. 4A′;see arrow). In vivo inner enamel epithelium also arranges in a columnarlayer during cap stage and gives then rise to pre-ameloblasts andameloblasts.

1. A method of preparing an artificial tooth primordium in vitro,comprising the steps: a) providing isolated mesenchymal dental pulpcells; and b) culturing the mesenchymal dental pulp cells undernon-adherent conditions to form a cell aggregate representing anartificial tooth primordium.
 2. The method of claim 1, wherein themesenchymal dental pulp cells comprise or consist of primary adultmesenchymal dental pulp cells.
 3. The method of claim 1, wherein themesenchymal dental pulp cells are derived from dental pulp tissue of atooth, preferably from pulp tissue of a third molar.
 4. The method ofclaim 1, wherein the mesenchymal dental pulp cells are subjected tonon-adherent culture conditions at a concentration of 5×10⁴ to 5×10⁷ perml.
 5. The method of claim 1, wherein the mesenchymal dental pulp cellsare cultured under non-adherent conditions for at least 48 hours.
 6. Themethod of claim 1, wherein the mesenchymal dental pulp cells arecultured under non-adherent conditions at least until a cell aggregateis formed with an average diameter of 0.3 mm to 2 mm.
 7. The method ofclaim 1, wherein the mesenchymal dental pulp cells are cultured undernon-adherent conditions at least until the cell aggregate formedexhibits upregulated expression of BMP4, HGF, PAX9, MSX1, Collagen typeI, DSPP and/or predentin compared to isolated mesenchymal dental pulpcells immediately prior to subjecting to non-adherent culturingconditions.
 8. The method of claim 1, wherein no artificial biologicalor non-biological scaffold is used in the formation of the cellaggregate.
 9. A method of preparing an artificial tooth primordium invitro, comprising the steps: a) providing isolated mesenchymal dentalpulp cells; and b) culturing the mesenchymal dental pulp cells undernon-adherent conditions to form a cell aggregate representing anartificial tooth primordium, wherein, after culturing of the mesenchymaldental pulp cells under non-adherent conditions for at least 2 days,epithelial cells are added and co-cultured under non-adherentconditions.
 10. The method of claim 9, wherein the epithelial cells arekeratinocytes, preferably keratinocytes derived from skin or gingiva.11. An artificial tooth primordium produced by a method of preparing anartificial tooth primordium in vitro, comprising the steps: a) providingisolated mesenchymal dental pulp cells; and b) culturing the mesenchymaldental pulp cells under non-adherent conditions to form a cell aggregaterepresenting an artificial tooth primordium.
 12. A transplant comprisingan artificial tooth primordium produced by a method of preparing anartificial tooth primordium in vitro, comprising the steps: a) providingisolated mesenchymal dental pulp cells; and b) culturing the mesenchymaldental pulp cells under non-adherent conditions to form a cell aggregaterepresenting an artificial tooth primordium.
 13. A pharmaceuticalcomposition comprising an artificial tooth primordium produced by amethod of preparing an artificial tooth primordium in vitro, comprisingthe steps: a) providing isolated mesenchymal dental pulp cells; and b)culturing the mesenchymal dental pulp cells under non-adherentconditions to form a cell aggregate representing an artificial toothprimordium and at least one pharmaceutically acceptable excipient.
 14. Amethod for screening substances in vitro, which modulate properties oftooth tissue, comprising the steps of: providing a sample of anartificial tooth primordium or a cell aggregate or tissue derivedtherefrom; wherein the artificial tooth primordium is produced by amethod of preparing an artificial tooth primordium in vitro, comprisingthe steps: a) providing isolated mesenchymal dental pulp cells; and b)culturing the mesenchymal dental pulp cells under non-adherentconditions to form a cell aggregate representing an artificial toothprimordium; dividing the respective sample into portions; incubating atleast one portion with a substance to be screened; and comparingparameters measured for the treated portion with another portion thatwas not incubated with the substance to be screened.
 15. The method ofclaim 14, wherein the portion is subjected to a self-containedcirculation system prior to incubating the portion with a substance tobe screened.